US7846909B2 - Method and compositions for inhibiting MAGE protein interaction with KAP-1 - Google Patents
Method and compositions for inhibiting MAGE protein interaction with KAP-1 Download PDFInfo
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Definitions
- CT antigens are a group of proteins originally defined by their normal expression in testes and their aberrant expression in melanomas and other cancers.
- the first CT antigens discovered were members of the MAGE family of proteins, including the MAGE-A, -B, and -C subfamilies which are encoded on the X-chromosome and which are now called Class I MAGE antigens (1, 2). Because many Class I MAGE genes are highly homologous and are co-regulated in gametogenesis and in tumors, it has been suggested that many MAGE proteins have similar or complimentary functions (3).
- MAGE gene expression can be caused by promoter region demethylation and is widespread in malignancies, being found in 50% or more of melanomas, synovial sarcomas, and primary carcinomas of the lung, head and neck, urinary bladder, and ovaries, as in well as lesser percentages of primary breast carcinomas and myelomas (5-8).
- KAP1 also known as TRIM28, Tif1 ⁇ , or Krip1
- TRIM28 TRIM28
- Tif1 ⁇ a RING-B-box coiled-coil domain near its amino terminal end (9-11).
- RBCC RING-B-box coiled-coil
- Complete loss of KAP1 function in the homozygous KAP1 knockout mouse is lethal in utero in the presence of functional p53, and KAP1 is increasingly being recognized as a central molecule in gene regulation (12). Binding to the RBCC motif of KAP1 is required for function of all KRAB domain containing zinc finger transcription factors (13, 14).
- KAP1 appears to function as a molecular scaffold that coordinates at least four activities necessary for gene specific silencing including: 1.
- KAP1 acts as a co-repressor of p53 by binding to MDM2, RING domain ubiquitin E3 ligase and a major repressor of the p53 tumor suppressor protein, thereby suppressing p53 expression, p53 acetylation and p53 function (15).
- Kap1 acts as a p53 co-repressor with MDM2 by several mechanisms including 1) increasing binding of MDM2 to p53; 2) contributing to MDM2 inactivation of p53 transcription functions; 3) increasing MDM2 inhibition of p53 acetylation) and 4) promoting MDM2 mediated ubiquitination and degradation of P53 (15).
- Preferable MAGE antagonists disclosed therein include an anti-MAGE antibody, an antisense molecule, an siRNA molecule, a molecule for forming a triplex nucleic acid molecule with a MAGE encoding polynucleotide, or a small molecule inhibitor of MAGE function.
- MAGE protein especially Class I MAGE protein
- expression suppresses apoptosis by suppressing p53 and actively contributes to the development of malignancies and promoting tumor survival
- inhibition of MAGE expression or function represents a novel and specific treatment for melanoma and diverse malignancies.
- MAGE proteins act as co-repressors of p53 by binding to KAP1 and enhancing its suppression of p53, and or the DNA damage response. MAGE proteins may contribute to the development of malignancies by providing a survival advantage and interfering with MAGE expression or function may prove to be a novel avenue for therapeutic intervention in a wide variety of other malignancies.
- MAGE-A, B, and C protein families comprise the Class-I MAGE/Cancer Testes Antigens, a group of highly homologous proteins whose expression is suppressed in all normal tissues except developing germ cells. Aberrant expression of Class I MAGE proteins occurs in melanomas and many other malignancies, and MAGE proteins have long been recognized as tumor specific targets, but their functions have largely been unknown.
- MAGE-A positive, p53 wt/wt parental HCT 116 colon cancer cells but not in a MAGE-A positive HCT 116 p53 ⁇ / ⁇ variant, indicating that p53 is involved in MAGE suppression of apoptosis.
- treatment with MAGE specific siRNA suppresses S91 melanoma growth in vivo, specifically in syngenic DBA2 mice.
- the present invention provides a method for inhibiting the growth or proliferation, or inducing apoptosis, of a mammalian cell that expresses a MAGE gene, the method comprising inhibiting the binding of KAP-1 to a polypeptide encoded by the MAGE gene in the cell.
- the method comprising administering to the cell a substance that inhibits the formation of a complex between KAP-1 and a MAGE protein, or the function of the complex.
- the substance may preferably be an antibody against a complex formed between KAP-1 and a polypeptide encoded by a MAGE gene.
- Such antibody may preferably be a monoclonal antibody, or an active fragment thereof, in particular a humanized or human antibody in the treatment of human cells, especially a cancerous or malignant or neoplastic cell.
- the method of the present invention comprises inhibiting MAGE function or expression in combination with one or more chemotherapeutic agent.
- the present invention further provides a method for regulating male fertility in a mammal, the method comprising inhibiting MAGE/KAP-1 formation in a male testis cell according to the method described herein.
- the present invention also provides a pharmaceutical composition for treating tumor cell formation, tumor cell growth, or induction of apoptosis of a testis cell comprising a compound that inhibits the binding of KAP-1 and a polypeptide encoded by a MAGE gene and a pharmaceutically acceptable excipient.
- the present invention provides a method for screening for a substance that inhibits MAGE/KAP-1 complex formation, the method comprising: (1) providing a candidate substance to be tested; (2) applying said candidate substance to a cell or protein array expressing a MAGE gene or MAGE gene construct, (3) detecting the level of MAGE/KAP-1 complex in the presence of the candidate substance, and (4) determining a level of MAGE/KAP-1 complex in the absence of the candidate substance, wherein a substance that decreases the level of MAGE/KAP-1 complex is selected to be an antagonist of MAGE/KAP-1 formation.
- FIG. 1 shows that MAGE siRNAs inhibit melanoma growth in vitro.
- MAGE siRNA inhibits the growth of human (Hs-294T, A375) and murine (S91) melanoma cell lines but not the MAGE ( ⁇ ) HaCaT human keratinocyte line. Trypan blue viability was determined 72 hours post transfection with 100 nM siRNA. The blue bar represents the growth of mock-transfected cells (no siRNA) and is considered as 100% viability.
- Pan-MAGE-A siRNA targets all human MAGE-A sub-family members except MAGE-A1, which has significant sequence variation which makes the use of a small common set of siRNAs impossible.
- Pan-mMage-a and mMage-b siRNAs target all murine mMage-a and mMage-b family members, respectively.
- the human melanoma cell lines do not express MAGE-B and there is no murine mMage-c. * Indicates a significant difference from non-specific siRNA (p ⁇ 0.05, T-Test). Error bars indicate s.d. Analyses were performed from at least 3 individual experiments with triplicates for each group.
- C. Individual siRNA duplexes targeting different sequences in mMage-b have variable results, indicating sequence specificity.
- NS non-specific control siRNA.
- D. MAGE siRNAs decrease expression of target proteins.
- NS non-specific control siRNA
- ⁇ no siRNA
- FIG. 3 shows that MAGE-A, mMage-b, and MAGE-C proteins form complexes with KAP1.
- Top blot immunoblotting with antibody against the C-terminal region of KAP1 shows that immunoprecipitation of MAGE-C2 only pulls down proteins containing the BB and Coiled-Coil regions (boxed bands indicate the full length KAP1 and the dR construct).
- Bottom blot Immunoblotting with antibody recognizing the N-terminal region of KAP1 shows that MAGE-C2 pulls down proteins lacking the C-terminal NHD, PHD and Bromo regions, (boxed bands indicate full length, dPB and RBCCH constructs).
- FIG. 4 shows that MAGE protein expression augments KAP1/p53 complex formation and suppression of p53.
- A Co-immunoprecipitation of endogenous KAP1 and p53.
- B The columns indicated with * show decreases in co-immunoprecipitated KAP1 and p53, and decreased MAGE protein in whole cell lysates after treatment with MAGE siRNA.
- C The histograms show the relative densities of the bands of co-immunoprecipitated p53 and Kap-1 shown in 4 B.
- FIG. 5 A MAGE siRNA suppresses the viability of the parental and p53RE-BLA HCT116 cell lines, but not in the p53 ⁇ / ⁇ variant of HCT116.
- B. MAGE-A knockdown activates a beta-lactamase reporter gene driven by a p53 consensus sequence promoter in the p53RE-BLA HCT116 cell line. Activation of the reporter gene indicates transcriptional activity of endogenous p53. The assays were performed at different time points post transfection as shown above, and p53 activity reached to peak 6 hours after MAGE gene was knockdown.
- C KAP1 siRNA inhibits growth of human melanoma cells. *p ⁇ 0.05, T-Test compared with non-specific siRNA treatment, this analysis was obtained from two individual experiments with triplicates for each group.
- FIG. 6 shows that MAGE siRNAs suppress the growth of melanoma in vivo.
- A Kaplan-Meier plot for mice injected with S91 cells transfected pre-inoculation with 100 nM control or mMage-b siSTABLE-PLUS siRNA.
- B Kaplan-Meier plot for mice inoculated with S91 cells then given intraperitoneal injections of mMage-b siSTABLE-PLUS siRNA, directly conjugated to cholesterol, on days 4, 6, 8, 10, and 11 after tumor inoculation.
- C. Validation study shows loss of mMage-b target mRNA in tumor cells 48 hours after mMage-b siRNA treatment.
- FIG. 7 shows real time RT-PCR quantification of murine mMage-a and mMage-b mRNA, indicating that mMage-b has higher expression than mMage-a at mRNA level in S91 cells.
- FIG. 8 shows the results of Tunnel Analysis, showing that MAGE siRNAs induced caspase-independent apoptosis in melanoma cells (TUNEL Analysis).
- Flow cytometry shows that apoptosis induced by MAGE siRNA in human Hs-294T (A) and murine S91 cells (B) is not inhibited by the general caspase inhibitor zVAD-FMK.
- Apoptotic cells are in upper, L-shaped window. The percentage of apoptotic cells is shown for each condition in the upper right corner of the individual panels.
- FIG. 9A shows that the yeast two hybrid assay, identifies MAGE-C2 as a Kap-1 binding partner.
- Yeast were co-transformed with KAP1 or control protein expression plasmids and grown on selection medium (left panel). Colonies were transferred and assayed for ⁇ -galactosidase activity (blue-green color, right panel).
- T7 yeast co-transformed with irrelevant bait, pGBK-T7;
- LaminC yeast co-transformed with irrelevant bait, pGBKT7-LaminC;
- KAP1 yeast co-transformed with pGBKT7-KAP1. Note that ⁇ -galactosidase activity was only seen in the presence of Kap-1, confirming Kap-1 MAGE-C2 binding.
- FIG. 9A shows that the yeast two hybrid assay, identifies MAGE-C2 as a Kap-1 binding partner.
- FIG. 10 depicts the result of immunoblotting which shows an increase in immunoreactive p53 in the nuclear fractions of the Hs-294T and A375 human melanoma cell lines 24 h after knockdown of MAGE-C2 compared to non-specific siRNA and no siRNA controls.
- Immunoblot of the nuclear protein Lamin B1 shows the purity of fractionation and serves as a protein loading control.
- FIG. 11 shows that MAGE-A protein is expressed in all three HCT 116 cell variants.
- FIG. 12 shows the result of an analysis of growth of mMage-b siRNA transfected S91 Cells in syngenic mice.
- A Linear regression analysis with tumor growth averaging 0.38 mm/d for nonspecific siRNA and 0.15 mm/d for mMage-b siRNA (p ⁇ 0.001).
- B C. Log-Rank analysis in Kaplan-Meier mean and median survival between non-specific and mMage-b siRNA. (p ⁇ 0.001).
- FIG. 13 depicts the result of an analysis of growth of S91 cells in syngenic mice treated with intraperitoneal injections of cholesterol conjugated siRNA.
- FIG. 14 shows that MAGE-A and MAGE-C2 siRNAs sensitize U226 Myeloma cells to the DNA damaging agents doxorubicin and paxlitaxol.
- FIG. 15 shows the siRNA sequences used herein.
- the present invention provides in one embodiment a method for screening for a substance that inhibits MAGE/KAP-1 complex formation.
- a method for screening for a substance that inhibits MAGE/KAP-1 complex formation To identify an inhibitor in accordance with the present invention, one generally will determine the ability of a candidate substance to inhibit MAGE protein-KAP-1 complex formation.
- the method may generally comprise (a) providing a MAGE protein or a relevant or functional fragment or subunit thereof a and KAP-1 under conditions permitting the formation of a complex, in the presence or absence of a candidate substance, and (b) assessing the formation of the complex, wherein a decrease in complex formation in the presence of a candidate substance, as compared to complex formation in the absence of the first substance, indicates that the candidate substance is an inhibitor of the MAGE protein and KAP-1 complex formation.
- Such assays can be performed in cell free environments, but also may be conducted in isolated cells, organs, or in living organisms, and may comprise random screening of large libraries of candidate substances.
- Candidate Substances refers to any molecule that inhibits under suitable conditions the formation of MAGE protein and KAP-1 complexes.
- Candidate compounds may be screened from large libraries of synthetic or naturally-occurring compounds, or fragments or parts thereof, which are readily available to those skilled in the art. It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents.
- the candidate substance may be a macromolecule such as a protein or fragment thereof (e.g. an antibody) or a nucleic acid molecule, and may be a small molecule.
- Initially identified compounds may serve as lead compounds to help develop improved compounds via “rational drug design,” via comparisons with known inhibitors, and modifications based on structural and other knowledge of the lead compound and further testing.
- the goal of rational drug design is to produce structural analogs or derivatives of biologically active compounds. By creating such analogs, it is possible to generate drugs which are more active or stable or otherwise more pharmaceutically desirable.
- a quick, inexpensive and easy assay to run is an in vitro assay.
- Such assays generally use isolated molecules, can be run quickly and in large numbers, thereby increasing the amount of information obtainable in a short period of time.
- isolated MAGE protein or a suitable functional fragment thereof are combined with isolated KAP-1 protein, and complex formation is assayed in the presence and absence of a candidate substance, under suitable conditions. The lack or reduction of complex formation in the presence of the candidate compound, as compared to in the absence of the candidate or another suitable control, indicates that the candidate substance is an inhibitor of MAGE/KAP-1 interaction or binding.
- the present invention may also utilize a variety of specific assay formats. Inhibition of protein-protein interactions may be studied by using biochemical and immunologic techniques, such as fluorescence energy transfer where different molecules are both labeled with appropriate fluorescent donor-acceptor pairs, co-immunoprecipitation, double-determinant Western blot and ELISAs (e.g., sandwich ELISA), which are well known to those skilled in the art.
- biochemical and immunologic techniques such as fluorescence energy transfer where different molecules are both labeled with appropriate fluorescent donor-acceptor pairs, co-immunoprecipitation, double-determinant Western blot and ELISAs (e.g., sandwich ELISA), which are well known to those skilled in the art.
- Fluorescence resonance energy transfer is a common technique when observing the interactions of two different proteins. Change in fluorescence intensity or spectrum is an indication of the binding of the two proteins, and can be used to assay the ability of a candidate substance to inhibit the complex formation.
- Label transfer can be used for screening or confirmation of protein interactions and can provide information about the interface where the interaction takes place. Label transfer can also detect weak or transient interactions that are difficult to capture using other in vitro detection strategies.
- a label transfer reaction a known protein is tagged with a detectable label. The label is then passed to an interacting protein, which can then be identified by the presence of the label.
- Tandem affinity purification detects interactions within the correct cellular environment (e.g. in the cytosol of a mammalian cell) (Rigaut et al., 1999, Nat. Biotechnol. 17:1030-2). This is a big advantage compared to the yeast two-hybrid approach. However, the TAP tag method requires two successive steps of protein purification. Thus, it can not readily detect transient protein-protein interactions.
- crosslinking is often used to “fix” protein interactions in place before trying to isolate/identify interacting proteins.
- Common crosslinkers for this application include the non-cleavable NHS-ester crosslinker, [[bis-sulfosuccinimidyl suberate]] (BS3); a cleavable version of BS3, dithiobis(sulfosuccinimidyl propionate) (DTSSP); and the imidoester crosslinker dimethyl dithiobispropionimidate (DTBP) that is popular for fixing interactions in ChIP assays.
- BS3 non-cleavable NHS-ester crosslinker
- DTSSP dithiobis(sulfosuccinimidyl propionate)
- DTBP imidoester crosslinker dimethyl dithiobispropionimidate
- Quantitative immunoprecipitation combined with knock-down relies on co-immunoprecipitation, quantitative mass spectrometry (SILAC) and RNA interference (RNAi).
- SILAC quantitative mass spectrometry
- RNAi RNA interference
- Dual Polarisation Interferometry can be used to measure protein-protein interactions.
- DPI provides real-time, high-resolution measurements of molecular size, density and mass. While tagging is not necessary, one of the protein species must be immobilized on the surface of a waveguide.
- Static Light Scattering measures changes in the Raleigh scattering of protein complexes in solution and can non-destructively characterize both weak and strong interactions without tagging or immobilization of the protein.
- the measurement consists of mixing a series of aliquots of different concentrations or compositions with the anylate, measuring the effect changes in light scattering as a result of the interaction, and fitting to a model. Weak, non-specific interactions are typically characterized via the second virial coefficient. This type of analysis can determine the equilibrium association constant for associated complexes (Attri et al., 2005, Analyt. Biochem. 346(1):132-138).
- the present invention also contemplates the screening of compounds for their ability to inhibit MAGE protein/KAP-1 complexes formation in cells.
- Various cell lines can be utilized for such screening assays, including cells specifically engineered for this purpose.
- genetic constructs for use in transforming cells for such assays may also be used.
- In vivo assays involve the use of various animal models of cancers with active or enhanced MAGE expression. Treatment of animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal. Administration will be by any route that could be utilized for clinical purposes. Determining the effectiveness of a compound in vivo may involve a variety of different criteria. Also, measuring toxicity and dose response can be performed in animals in a more meaningful fashion than in vitro or in cyto assays.
- a yeast or mammalian two-hybrid screen See: for example, U.S. Pat. No. 5,283,173
- the system uses a native MAGE protein or a variant thereof, and KAP-1 or a variant thereof, fusion proteins, where MAGE is fused to the DNA binding domain and KAP-1 is fused to the activation domain.
- the assay could also be configured with MAGE fused to the activation domain and KAP-1 fused to the DNA binding domain.
- Compounds are preferably selected based on their ability to block (or inhibit) yeast growth or reporter gene expression in yeast or mammalian cells.
- Preferred reporter gene includes those encoding ⁇ -galactosidase synthesis (blue color), chloramphenicol acetyltransferase (CAT), luciferase, or other fluorescent proteins (e.g. GFP).
- the yeast cell is permeabilized (Gaber, et al., Mol. Cell. Biol. 9: 3447-3456 (1989)). It is appreciated that certain modification to the basic dihybrid screen may be desirable, such as, substituting other DNA binding and activation domains for those contributed by GAL-4 (e.g., lexA DNA binding domain is also contemplated for use in this invention.
- inducible promoters can be used to drive expression in the two hybrid screens of this invention.
- Two-hybrid based screening method of the present invention for MAGE/KAP-1 binding inhibitors may be devised using a high-throughput, microtiter-formatted, robotics-amenable system.
- Candidate compounds may be seeded individually at a suitable concentration (e.g. 25 ⁇ g/ml) with 2% galactose to induce yeast protein expression and hybrid formation.
- Yeast or mammalian cells pre-grown in selective media containing the neutral carbon source raffinose are added, and the increase in candidate gene expression such as beta-gal activity (yeast two hybrid method) or chloramphenicol acetyl transferase (CAT) or Luciferase (mammalian two hybrid methods) are measured following incubation.
- beta-gal activity yeast two hybrid method
- CAT chloramphenicol acetyl transferase
- Luciferase mimmalian two hybrid methods
- Percent inhibition may be calculated, and a differential value determined with respect to a parallel control two-hybrid strain.
- the control strain should be the same parent yeast strain or cell line containing plasmids encoding the two proteins without any addition of the candidate compounds, or two other proteins that are known to interact.
- Modification to the above may include, but are not limited to, different yeast strains or transfectants with different transcriptional reporters (i.e. auxotrophic markers like URA3), a different two-hybrid system (Fields, et al., (1989) Nature 340:245-247), and different promoters on the plasmids.
- different transcriptional reporters i.e. auxotrophic markers like URA3
- two-hybrid system Yields, et al., (1989) Nature 340:245-247
- promoters on the plasmids may include, but are not limited to, different yeast strains or transfectants with different transcriptional reporters (i.e. auxotrophic markers like URA3), a different two-hybrid system (Fields, et al., (1989) Nature 340:245-247), and different promoters on the plasmids.
- a non-transfected cell based assay may be used for screening for antagonists of KAP-1/MAGE binding. This would allow the identification of antagonists that would function inside the cell. Specifically, a MAGE positive cell line is cultivated in the presence and absence of a candidate compound, and the viability of the cells are measured. Compounds that cause the cells to lose viability are identified, and their ability to inhibit MAGE/KAP-1 binding confirmed by assaying the cells cultured in the presence of these compounds for the absence of MAGE/KAP-1 complex, e.g. by immuno-precipitation assays.
- p53 level and/or ubiquitination and degradation by immunoblotting instead of measuring cell viability/growth rate, p53 level and/or ubiquitination and degradation by immunoblotting, p53 acetylation, over even p53 transcription, p53 function, and recruitment of histone deacetylases may be assayed as an indication that KAP-1/MAGEbinding has been inhibited, followed by confirmation of the lack of KAP-1/MAGE complex by e.g. immuno-precipitation assays.
- Inhibitors can be discovered directly in vitro with purified MAGE and KAP-1. If in the presence of a candidate compound a MAGE/KAP-1 complex fails to form in vitro, while in the absence of the candidate the complex forms under otherwise identical conditions, then the candidate compound would be considered an inhibitor of MAGE and KAP-1 binding.
- an assay There are many ways to configure an assay. For example, a 96-well plate in which one of the binding partners, preferably purified recombinant proteins, is initially added to the wells of an ELISA plate, followed by an additional incubation with a non-specific protein such as BSA (bovine serum albumin) to block free binding sites on the plastic. Subsequently a solution containing the putative inhibitor or control buffer is added mixed with a solution containing the other binding partner and incubated so as to allow complete binding of MAGE to KAP-1 in the control buffer well. The complex so formed in each well is then measured. This may be accomplished e.g. by either labeling one of the binding partners and then detect the complex directly, e.g. by electrophoresis, or detect the complex using an antibody specific for the complex.
- BSA bovine serum albumin
- the present invention provides a method for inhibiting the growth or proliferation, or inducing apoptosis, of a cell that expresses a MAGE gene, the method comprising inhibiting the formation MAGE gene product with KAP-1, thereby inhibiting the function the MAGE gene in the cell.
- Suitable MAGE genes for the present invention may be a Type I MAGE gene, such a MAGE-A, MAGE-B, or MAGE-C, specifically, MAGE, A3, A5, A6, A8, A9, A10, A11 or A12, or MAGE-B1, B2, B3 or B4, or MAGE-C1, or C2.
- the MAGE gene may also be a Type II MAGE gene, such as Necdin, MAGE-D, MAGE-E (E1), MAGE-F, MAGE-G, or MAGE-H.
- the method inhibits MAGE gene function in a cell A which is a cancerous or malignant or neoplastic cell.
- the cancer, tumor, or cellular proliferation is selected from the group consisting of melanomas, Multiple Myeloma and other plasma cell dyscrasias, lymphoma, T-cell leukemia, non-small cell lung carcinoma, hepatic carcinoma, gastric cancer, esophagus carcinomas, colorectal carcinomas, pancreatic endocrine neoplasms, ovarian neoplasms, cervical cancer, salivary glands carcinoma, head and neck squamous cell carcinomas, proliferating testes cells, spermatocytic seminoma, sporadic medullary thyroid carcinoma, osteosarcomas, childhood astrocytomas, bladder cancer, cells from inflamed joints in juvenile rheumatoid arthritis or other harmful inflammatory condition, glioma, neuroblastoma tumor, or other harmful inflammatory condition,
- the present invention provides antagonist compositions and methods that inhibit MAGE protein from binding to KAP-1.
- an embodiment of the present invention provides contraceptive methods and compositions for male mammals, especially in man.
- the contraceptive methods comprise administering to the mammal in need thereof a composition comprising one or more antagonists of MAGE protein/KAP-1 binding, such as a small molecule antagonist, siRNA, an antibody against a MAGE gene product, or an antisense nucleic acid molecule.
- the antagonists of the invention are preferably used as a treatment for cancer formation or growth.
- neutralizing it shall be understood that the antagonist has the ability to inhibit or block the binding of MAGE to KAP-1.
- An anti-MAGE antibody suitable for the present invention may be a polyclonal antibody.
- the antibody is a monoclonal antibody.
- the antibody may also be isoform-specific.
- the monoclonal antibody or binding fragment thereof of the invention may be Fab fragments, F(ab) 2 fragments, Fab′ fragments, F(ab′) 2 fragments, Fd fragments, Fd′ fragments or Fv fragments.
- Domain antibodies dAbs
- antibodies with a known antigen are well-known to those ordinarily skilled in the art (see for example, Harlow and Lane, 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; see also WO 01/25437).
- suitable antibodies may be produced by chemical synthesis, by intracellular immunization (i.e., intrabody technology), or preferably, by recombinant expression techniques.
- Methods of producing antibodies may further include the hybridoma technology well-known in the art.
- the antibodies or binding fragments thereof may be characterized as those which are capable of specific binding to a MAGE protein/KAP-1 complex or an antigenic fragment thereof, preferably an epitope that is recognized by an antibody when the antibody is administered in vivo.
- Antibodies can be elicited in an animal host by immunization with a MAGE/KAP-1 complex-derived immunogenic component, or can be formed by in vitro immunization (sensitization) of immune cells.
- the antibodies can also be produced in recombinant systems in which the appropriate cell lines are transformed, transfected, infected or transduced with appropriate antibody-encoding DNA. Alternatively, the antibodies can be constructed by biochemical reconstitution of purified heavy and light chains.
- the antibodies may be from humans, or from animals other than humans, preferably mammals, such as rat, mouse, guinea pig, rabbit, goat, sheep, and pig. Preferred are mouse monoclonal antibodies and antigen-binding fragments or portions thereof.
- chimeric antibodies and hybrid antibodies are embraced by the present invention. Techniques for the production of chimeric antibodies are described in e.g. Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; and Takeda et al., 1985, Nature, 314:452-454.
- single chain antibodies are also suitable for the present invention (e.g., U.S. Pat. Nos. 5,476,786 and 5,132,405 to Huston; Huston et al., 1988, Proc. Natl. Acad. Sci. USA, 85:5879-5883; U.S. Pat. No. 4,946,778 to Ladner et al.; Bird, 1988, Science, 242:423-426 and Ward et al., 1989, Nature, 334:544-546).
- Single chain antibodies are formed by linking the heavy and light immunoglobulin chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Univalent antibodies are also embraced by the present invention.
- liposomes with antibodies in their membranes to specifically deliver the liposome to the area of the tumor where MAGE expression or function is to be inhibited.
- liposomes can be produced such that they contain, in addition to monoclonal antibody, other therapeutic agents, such as those described above, which would then be released at the tumor site (e.g., Wolff et al., 1984, Biochem. Biophys. Acta, 802:259).
- Hs-294T, A375 and S91 melanoma cell lines were purchased from ATCC (Manassas, Va.).
- p53RE-bla HCT-116 cell was purchased from Invitrogen (Invitrogen, Carlsbad, Calif.), and HCT 116 p53 wt/wt or p53 ⁇ / ⁇ cell lines were kindly provided by Bert Vogelstein (The Johns Hopkins University School of Medicine, Baltimore, Md.).
- the HMC1 malignant mast cell line was kindly provided by Dr. J. H. Butterfield, The Mayo Clinic, Rochester, Minn. (18).
- siRNA All siRNA preparations were siStable or siSTABLE-Plus (conjugated to cholesterol at the 5′ end of the sense strand) siRNA purchased from Dharmacon Inc., Boulder, Colo. In some in vivo studies we used PEI complexed siRNA (JET-PEI, PolyPlus Transfection, Illkirch, France). The specific targets for each individual siRNAs were described in Yang et al (17). Sequences of siRNA sequences are given in FIG. 14 .
- Antibodies For human target validation studies and immunoprecipitation we used: pan-MAGE-A monoclonal (Zymed laboratories Inc, South San Francisco, Calif.), anti-MAGE-A1 monoclonal (Santa Cruz, Santa Cruz, Calif.), anti-MAGE-B2 (polyclonal, Santa Cruz, Santa Cruz, Calif.) or anti-human MAGE-C2 monoclonal (Ludwig Institute for Cancer Research, New York, N.Y.), anti-human KAP1 (polyclonal, Novus, Biologicals, Littleton, Colo., recognizing the N-terminal region 1-50 amino acids of KAP1), anti-human KAP1 (polyclonal, supplied by Dr. Frank J.
- V5-Tagged mMage-b was expressed using pcDNA3.1/V5-TOPO vector from Invitrogen (19).
- V5-Tagged MAGE-A3 was expressed using the T-Rex viral power lentivirus system (Invitrogen).
- FLAG-Tagged MAGE-C2 was expressed using pFLAG-CMV-2 (Sigma).
- FLAG-Tagged KAP1 cDNAs with various deletions were made in the Rauscher lab and expressed using pcDNA3.1.
- siRNA Transfection and Cell Viability In vitro studies used LipofectamineTM 2000 (Invitrogen, Carlsbad, Calif.) as a transfection reagent and all transfections were performed under a Rnase-free condition. The final siRNA concentrations were 50, 100 or 150 nM. Cell viability was determined 72 hours after transfection by counting cells that excluded trypan blue. In selected experiments, cell viability was also determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Sigma, St. Louis, Mo.) which showed similar results to the Trypan Blue assay (data not shown).
- Target Validation and Specificity Confirmation of cleavage of mRNA induced by siRNA was previously documented (17). Target protein suppression was validated by immunoblotting forty-eight hours post transfection ( FIG. 1D ).
- Apoptosis Assays Apoptosis was determined by morphologic analysis following staining with acridine orange and ethidium bromide (17), or by APO-BrdUTM TUNEL assay (Molecular Probes, Eugene, Oreg.). Both assays were performed approximately one half doubling time after transfection with a final siRNA concentration of 100 nM.
- MAGE-C2 The human MAGE-C2 (NM — 016249, previously called MAGE-E1) was used as a bait protein and the Clontech YEASTMAKER kit was used to construct a cDNA library from mRNA of the HMC1.1 malignant mast cell line. 1.2 ⁇ 10 6 independent transformants were obtained, yielding 86 colonies, of which 51 showed ⁇ -galactosidase activity and were sequenced. KAP1 (Trim 28) was identified as a potential partner of MAGE-C2.
- COS-7 cells were co-transfected with MAGE-C2 or V-5-mMage-b expression plasmids and with plasmids expressing full or partial length KAP1. Lysates were immunoprecipitated with anti MAGE-C2 or V5 antibody and blotted with antibodies recognizing either the N- or the C-terminal region of KAP1.
- P53 and acetylated p53 Cytoblots To determine the amount of acetylated p53 and total p53 levels in cells treated with siRNA we used the cytoblot technique (20). Cells are plated at 20,000 cells per well using a uFill reagent dispenser (Biotek Instruments, Inc.), allowed to attach overnight, and treated with siRNA. Following treatment, the cells are fixed by addition of 100 ul of 3.7% formaldehyde using a Biomek FX liquid handler (Beckman Coulter, Inc.).
- the plates are incubated for 20 minutes at 4 degrees and cells are washed 5 ⁇ in 100 ul of PBS pH 7.4 containing 0.1% Triton X-100 using a Biomek FX liquid handler to permeabilize cell membranes. Following permeabilization, cells are incubated in 100 uL/well of Odyssey Blocking Buffer (Licor, Inc.) for 1 hour at room temperature with gentle rotation.
- Odyssey Blocking Buffer Lior, Inc.
- Each well is incubated with 100 uL per well anti-p53 antibodies (Cell Signaling Technologies) at a 1:1000 dilution in Odyssey Blocking Buffer for one hour, followed by incubation with anti-p53 or anti-acetylated p53 antibodies (Cell Signaling Technologies) at a 1:1000 dilution in Odyssey Blocking Buffer for one hour.
- Secondary antibody incubations are carried out simultaneously by addition of 100 uL per well containing 1:5000 dilution of 680 nM dye conjugated anti mouse secondary antibody (Licor, Inc.) and 1:5000 dilution of 800 nM dye conjugated anti-rabbit secondary antibody (Licor, Inc.) for 1 hour.
- P53 activation assay To determine if MAGE gene knockdown affects the activity of p53, we utilized Invitrogen's GeneBLAzer® cell signaling pathway specific CellSensor® cell line, HCT-116 p53-BLA containing the GeneBLAzer® betalactamase (bla) Reporter Technology. When the p53 pathway is activated or inhibited, beta-lactamase reporter activity is modulated and can be measured quantitatively and selectively with the LiveBLAzerTM-FRET B/G Loading Substrate (InVitrogen). 12,000 cells per well are plated in each well of 384 well microplates using a uFill Reagent Dispenser (BioTek Instruments, Inc.).
- FRET B/G Loading Substrate InVitrogen
- the substrate molecule remains intact.
- excitation of the coumarin results in fluorescence resonance energy transfer to the fluorescein moiety and emission of green light.
- the substrate is cleaved, separating the fluorophores, and disrupting energy transfer. Excitation of the coumarin in the presence of enzyme bla activity results in a blue fluorescence signal.
- Beta lactamase assays were read in a Safire II microplate reader (Tecan, Inc.) at excitation 409 and emissions 460 nM and 535 nM.
- S91 murine melanoma cells were injected subcutaneously into the flanks of syngenic DBA/2 mice.
- cells were transfected with 100 nM siSTABLE-PLUS mMage-b siRNA or 100 nM control siSTABLE-PLUS siRNA in LipofectamineTM 2000 prior to inoculation. After 8 hours, equal numbers of viable cells were injected subcutaneously into the flanks of DBA/2 mice and tumor growth was followed as described (17).
- untreated S91 cells were injected subcutaneously on day “0” and then the mice were treated with multiple intra-tumoral or intraperitoneal injections of stabilized siRNA complexed with PEI or conjugated to cholesterol.
- FIG. 1 Working with human melanoma cell lines that express MAGE-A and MAGE-C2 proteins, and with a murine melanoma cell line that expresses mMage-b, we found that transfection with siRNAs targeting several MAGE genes decreased cell viability compared to the same cells transfected with control siRNA ( FIG. 1 ). Because of the high degree of homology of many MAGE family members and the lack of antibodies recognizing specific MAGE family members, we used siRNAs that target whole sub-families as well as siRNAs that target individual MAGE genes. FIG.
- FIG. 1A shows significant growth inhibition of human melanoma cells by siRNAs targeting common regions of the human MAGE-A gene family or targeting the human MAGE-C2 gene, but no significant effect with siRNA targeting the MAGE-B family members, which are not significantly expressed by these cells ( FIG. 1D ).
- mMage-b siRNA inhibits the viability of the murine S91 cell line, which expresses significantly more mMage-b than mMage-a ( FIG. 7 ).
- FIG. 1B shows siRNAs targeting unique sequences in several individual MAGE-A family members also effectively inhibit cell viability, except for MAGE-A1, which differs significantly from the other MAGE-A family members.
- FIG. 1C shows individual siRNA duplexes targeting different sequences in mMage-b have variable effects, indicating sequence specificity. Note that these siRNA reagents are species specific and have been previously shown to specifically destroy their target mRNAs (17). FIG. 1D shows that the siRNAs specifically suppress the targeted MAGE proteins.
- KAP1 is a Binding Partner of Multiple MAGE Proteins
- KAP1 is known to co-repress p53 by several mechanisms including decreasing p53 expression by facilitating its degradation and by blocking p53 acetylation and DNA binding (transcriptional activating) function (15). Therefore, we next looked for interactions between p53, MAGE, and KAP1. Unlike Monte et al, we did not detect direct binding of MAGE proteins to p53 (data not shown). However, we did find that KAP1 formed complexes with p53 and that MAGE knockdown decreased KAP1/p53 binding ( FIG. 4A , B, C). MAGE knockdown also resulted in increased immunoreactive p53 and acetylated p53 ( FIG. 4D and FIG.
- MAGE knockdown could not induce apoptosis in the absence of p53, since it has no significant effect on viability or apoptosis of HCT116 cells that are p53 ⁇ / ⁇ .
- MAGE knockdown activated an integrated beta lactamase reporter gene controlled by a consensus p53 responsive element in the p53RE-BLA variant of the HCT116 cell line ( FIG. 5B ).
- knockdown of KAP1 should also decrease cell viability.
- KAP1 knockdown did indeed suppress cell viability ( FIG. 5C ).
- our data allow us to conclude that expression of select Class I MAGE proteins promotes cell viability by preventing apoptosis and that a likely mechanism of action is that MAGE proteins function as co-factors supporting KAP1 dependent suppression of p53.
- FIG. 6A shows by Kaplan-Meier plot that pre-treatment with mMage-b siRNA significantly suppresses tumor growth and improves survival. (Please see FIG. 12 for additional analysis). Furthermore, although all control siRNA treated mice developed tumors, three of the mice receiving mMage-b siRNA treated cells never developed tumors.
- the knockdown studies disclosed herein fulfill criteria for demonstrating a “classical” RNAi response including showing specificity of reagents and reduction of gene expression at the mRNA and protein levels (20).
- the studies of individual siRNAs targeted to different mMage-b mRNA sequences serve as multiplicity controls by demonstrating similar biologic effects with two or more siRNAs, and together with the irrelevant control siRNAs and cross species studies indicate the sequence specificity of induction of apoptosis.
- the anti-MAGE-A antibody used for immunoprecipitation recognizes a common antigen present on all MAGE-A proteins except MAGE-A1. Although it is not clear whether all of the individual MAGE-A proteins interact with KAP1 in the cell lines used, the data on cell viability using siRNAs specific for individual MAGE-A family members suggest that suppression of apoptosis is a common function of multiple MAGE molecules, and that this may be a function of binding between the MAGE common homology domain and the KAP1 BB-CC region. The above data also support a previous proposal that the existence of multiple nearly identical MAGE family members enables a single critical function to be expressed under different transcriptional controls (1).
- MAGE-A1 appears to be an exception since suppression of MAGE-A1 does not significantly reduce cell viability.
- MAGE-A1 has the least common homology among the MAGE-A proteins and unlike other Class I MAGE proteins has been shown to bind to and inhibit the activity of the intracellular portion of Notch1, a SKIP-interacting transactivator (23).
- Notch1 a SKIP-interacting transactivator
- MAGE proteins were the first CT antigens discovered, and their limited tissue distribution has long been recognized as a potential key to tumor specific treatment of many different malignancies (2).
- MAGE genes are normally expressed in cells of the spermatogenic series during meiosis suggests that they may be members of the family of germ line anti-apoptotic genes which are involved in the maintenance of genomic stability and fertility in mammalian germ cells, and may protect cells from triggering an apoptotic response during meiosis (37, 38).
- MAGE siRNA decreases U226 Myeloma cell growth and synergizes with doxorubicin and paxlitaxol to decrease viability as determined by Trypan Blue exclusion at 72 h.
- U226 Myeloma cell cultures were started with 400,000 cells per well, were treated with MAGE-A3 and MAGE-C2 siRNA, with or without doxorubicin (10 nmol/L) or placlitaxel (1 nmol/L). The results are shown in FIG. 14 . These data show that treatment with MAGE-A3 and MAGE-C2 siRNA decreases the viability of the U226 Myeloma cells and results in increased cell death when combined with doxorubicin and paxlitaxol. In wells treated with MAGE siRNA and doxorubicin or paxlitaxol, cell numbers actually decreased. In the figure, * denotes values that are significantly different from comparable drug and culture conditions with control siRNA by T-test.
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Abstract
Description
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